Melanoma is one of the fastest-rising malignancies in the past few decades. It is a serious form of cancer responsible for approximately 80% of skin cancer-related deaths. Melanoma is resistant to current therapeutic regimens because for its propensity to metastasize and has a very poor prognosis with a median survival rate of six months. Studies have demonstrated that the majority of human melanomas have constitutively active mitogen activated protein kinases (MAPK) through oncogenic mutation in either BRAF or NRAS. In malignant melanoma the BRAF-MEK-ERK and PI3K-AKT-NFkB signaling pathways are constitutively activated and regulated by RAS signals. Activation of these pathways leads to an induction of epithelial-mesenchymal transition (EMT) resulting in cell invasion. EMT is a molecular program whereby epithelial cells undergo reprogramming from a polarized differentiated phenotype to a mesenchymal phenotype and is considered to be a key process in regulating the initial step of metastatic progression. Targeting BRAF with sorafenib resulted in inhibition of MAPK signaling pathways both in vitro and in vivo. However, as a monotherapy it is not effective in patients with metastatic melanoma as revealed by a phase II clinical study. These data and other published studies support the hypothesis that it is not sufficient to inhibit only a single constitutively activated signaling pathway for the treatment of melanoma. Recently, we found that fisetin treatment reduced melanoma cell invasion by inhibiting the protein expression of PI3K (p110a and p85) and phosphorylation of AKT at Ser473 and Thr308. It also reduced invasive potential of metastatic melanoma A375 cells in tissue-engineered three dimensional skin equivalents. Furthermore, fisetin treatment was found to reduce the formation of A375 cell colonies. The present proposal capitalizes on our recent novel unpublished findings and is designed to investigate the effects of fisetin alone and in combination with sorafenib on invasion and metastasis of melanoma cells by targeting BRAF and PI3K/AKT pathways. The central hypothesis to be tested in this proposal is that the combination of fisetin with sorafenib will be more effective in reducing melanoma cell invasion and metastasis than with fisetin or sorafenib alone. To test our hypothesis, two inter-related specific aims are proposed: (I) to investigate the effect of fisetin alone and in combination with sorafenib on invasion and EMT in melanoma cell lines and in tissue-engineered three dimensional skin equivalents by targeting BRAF and PI3K/AKT signaling pathways, and (II) to determine the effect of fisetin alone and in combination with sorafenib on the formation of systemic metastases in athymic Crl:NU-Foxn1nu nude mice by an intravenous injection of GFP-tagged melanoma cells. HEALTH RELEVANCE: Through this proposal we envision the identification of a natural nontoxic dietary flavonoid fisetin, which can be exploited in combination with sorafenib for the prevention of cell invasion and metastasis from which a large number of patients would stand to benefit.